Proteins are important as these are used to cure a number of diseases including angiogenesis (VEGF) diabetes (e.g. Insulin), cancers (e.g. Interferon, monoclonal antibodies), heart attacks, strokes, cystic fibrosis (e.g. Enzymes, Blood factors), inflammation diseases (e.g. Tumor Necrosis Factors), anemia (e.g. Erythropoietin), hemophilia (e.g. Blood clotting factors) etc. One of the important challenges is the development of efficient and competent process for the cloning, expression and large scale purification of these proteins. Numerous processes are available for the cloning, expression and large scale purification of desired protein from the cell culture supernatants, but still it is difficult to clone, express and separate the desired protein from the mixture.
Vascular endothelial growth factor A (VEGF-A) is a biological component that can trigger angiogenesis, which is the growth of new blood vessels. Various diseases, inter alia, ischemia, anemia, peripheral vascular disease, and atherosclerotic lesions can be treated by increasing angiogenesis. This is accomplished by stimulating the up-regulation of VEGF-A, thereby leading to increased blood circulation, hence increased oxygen supply, in the diseased tissue. In the eye, however, excessive vascularization can result in blood and fluid leaking into the eye. These leaky blood vessels can contribute to macular edema and choroidal neovascularization, resulting in the wet type of age-related macular degeneration (AMD). The result of AMD can be the loss of visual acuity or even blindness. Therefore, control of excessive macular vascularization is important in the treatment of macular degeneration. As such, it is a goal of medical professionals to provide a treatment for controlling or curing AMD without inhibiting the beneficial effects of normal VEGF-A activity in the rest of the body. Ranibizumab has been found to be an effective treatment of AMD.
Ranibizumab is a recombinant humanized IgGI kappa isotype monoclonal antibody that inhibits VEGF activity by competitively binding to the receptor binding site of active forms of VEGF-A, including the biologically active, cleaved form of this molecule, VEGF110. Hence, Ranibizumab prevents binding of VEGF-A to its principle receptors VEGFR1 and VEGFR2 found on the surface of endothelial cells. This results in reduced endothelial cell proliferation, vascular leakage, and new blood vessel formation.
LUCENTIS® is a medical formulation of Ranibizumab and designed for intraocular injection directly into the vitreous humor of the eye, wherein the active ingredient Ranibizumab penetrates the internal limiting membrane to access the subretinal space. These injections are typically given from 5 to 7 times a year to patients and in many instances are given monthly. Since the cost of treatment is very high and not many patients can afford it as such, there is still a need for improved cloning process and purification of the protein so that the production efficiency can be enhanced and the drug can be produced cheaper.
U.S. Pat. No. 6,998,383 discloses RANK inhibitor consisting of a TRAF-6 binding domain attached to a leader sequence.
U.S. Pat. No. 7,569,384 discloses nucleic acid molecule encoding albumin fusion protein. Also discloses vectors containing these nucleic acids, host cell transformed with these nucleic acid vectors and methods of making the albumin fusion protein using these nucleic acids, vectors and/or host cell.
U.S. Pat. No. 8,597,907 discloses a method for efficiently producing an industrially useful protein in coryneform bacteria. The present invention provides a method for efficiently producing heterologous proteins comprising culturing coryneform bacteria containing an genetic construction containing a promoter sequence which functions in coryneform bacteria, a nucleic acid sequence encoding a Tat system-dependent signal peptide region, and a nucleic acid sequence encoding a heterologous protein, in the direction from 5′-end to 3′-end, and secretory producing the heterologous protein by coryneform bacteria.
U.S. Pat. No. 5,641,870 discloses a process for purifying an antibody. In this process, a mixture containing the antibody and contaminant is subjected to low pH hydrophobic interaction chromatography (LPHIC) optionally at low salt concentration. The antibody is eluted from the column in the fraction which does not bind thereto. This process can be preceded and followed by other purification steps.
U.S. Pat. No. 7,847,071 discloses a method of purifying an antibody comprising the steps of: firstly, purifying an antibody by means of protein A affinity chromatography wherein the protein A is a native protein A or a functional derivative thereof, secondly, loading the purified antibody comprising a protein A-contaminant, wherein said protein A-contaminant is obtained upon eluting bound antibody from said protein A affinity chromatography, on a first ion exchanger under conditions which allow for binding of the protein A or its derivative, thirdly, collecting the antibody loaded onto the first ion exchanger in the flow-through of the first ion exchanger whilst a contaminant protein A is bound to the first ion exchanger, wherein the first ion exchanger is an anion exchanger, further purifying the antibody by loading on, binding to and eluting it from a second ion exchanger, and discarding a tail fraction of the eluate of the second ion exchanger such that a monomeric antibody fraction is enriched as a purified antibody pool.
WO2011089212 discloses a method for depleting impurities, in particular host cell proteins (HCP) and DNA from cell culture supernatants by means of protein A chromatography using a novel washing buffer.
Liu et al., discussed in mAbs 2, 2010, 480-499 about the basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps along with alternative processes such as flocculation, precipitation and membrane chromatography and also covered platform approaches to purification methods development, use of high throughput screening methods, and offered a view on future developments in purification methodology as applied to monoclonal antibodies.
Refolding of inclusion body proteins into bioactive forms is cumbersome, requires many operational steps and most of the time results in very low recovery of refolded protein. In the cases where a high yielding recovery process has been developed for refolding of the aggregated protein, inclusion body formation provides a straight forward strategy for recombinant protein purification. The higher the amount of this partially folded protein that is converted into the bioactive form, the more therapeutic protein can be recovered with improved yield and at low cost from inclusion bodies of E. coli. 
It is necessary to have a method for the cloning, expression and purification of antibodies and preferably without cost-intensive chromatographic steps as well as extensive steps. The antibody obtained by the cloning, expression and purification method according to the present invention is supposed to meet the criteria for purity & yield which are set forth by the admission authorities.